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Endocrinology Vol. 144, No. 9 4180-4186
Copyright © 2003 by The Endocrine Society

Expression of Bambi Is Widespread in Juvenile and Adult Rat Tissues and Is Regulated in Male Germ Cells

Kate Lakoski Loveland, Marilyn Bakker, Terri Meehan, Elizabeth Christy, Viktoria von Schönfeldt, Ann Drummond and David de Kretser

Monash Institute of Reproduction and Development, Monash University (K.L.L., M.B., T.M., E.C., V.v.S., D.d.K.), and Prince Henry’s Institute for Medical Research (A.D.), Melbourne 3168, Australia; and Institute of Reproductive Medicine of the University (V.v.S.), Munster 48149, Germany

Address all correspondence and requests for reprints to: Dr. Kate Loveland, Monash Institute of Reproduction and Development, 27-31 Wright Street, Clayton, Victoria 3168, Australia. E-mail: kate.loveland{at}med.monash.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Members of the TGFß superfamily may compete for receptor occupancy and intracellular signaling molecules in specific developmental circumstances. We explored the potential importance of the TGFß family inhibitor, Bambi (Bmp and activin membrane-bound inhibitor) by examining its pattern of mRNA expression in juvenile and adult rat tissues, with a focus on reproductive organs. The 1.8-kb transcript was ubiquitous, whereas a 3-kb transcript was unique to enriched spermatocyte and spermatid cell fractions and adult testis. The full-length rat cDNA is 89% (nucleic acid) and 95% (amino acid) identical to its human homolog, hnma. Using in situ hybridization, Bambi mRNA was detected in granulosa and thecal cells of adult ovaries and in spermatogonia, spermatocytes, round spermatids, and Sertoli cells of adult testes. In addition to a persistent signal in Sertoli cells in juvenile testes, this mRNA within germ cells appeared dramatically increased as gonocytes matured into spermatogonia immediately after birth. These data indicate that TGFß superfamily signaling within male germ cells is down-regulated at the onset of spermatogenesis. The addition of exogenous activin A to 24-h cultures of newborn rat testis fragments decreased the Bambi mRNA level. Regulated Bambi mRNA synthesis may contribute to TGFß superfamily signaling modulation in several organs, as suggested by its discrete expression switch in male germ cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
REGULATION OF TGFß superfamily signaling is known to occur through 1) extracellular ligand sequestration and 2) modulated expression of the interacting intracellular Smad signaling molecules (reviewed in Ref.1). A new regulatory mechanism was recently uncovered when the Xenopus homolog of hnma, named Bambi (Bmp and activin membrane-bound inhibitor), was described in Xenopus as a TGFß superfamily type I receptor subunit lacking the intracellular kinase domain required for signaling (2). hnma was originally identified in an mRNA subtractive hybridization screen designed to identify genes associated with a change in the metastatic potential of human melanoma cell lines (3). In experiments performed with its Xenopus and Danio homologs, coexpression of Bambi with other TGFß receptor subunits blocked signaling by TGFß, activin, and bone morphogenetic protein (BMP), and the protein was shown to form complexes with type 1 and 2 receptor subunits (1, 4).

Several TGFß superfamily members have been implicated in the regulation of mammalian gonadal development. In the mouse fetus, in vivo expression of BMP4 in the extraembryonic ectoderm is required to establish the primordial germ cell population before gonadal sex determination (5). After their differentiation and proliferation in the fetal rat testis, the quiescent gonocytes present in the last days of fetal life and immediately after birth contain both TGFß1 and -ß3 ligand subunits and TGFß type I and type II receptor subunits (6). In organ cultures, neither TGFß1 nor TGFß2 had a discernable impact on quiescent fetal rat germ cells. However, they each increased gonocyte apoptosis in fragments of the d 3 postpartum testis (3 dpp), coincident with the time when these male germ cells reenter the cell cycle and migrate to the perimeter of the seminiferous cord (7). We recently described the discrete down-regulation of activin ßA-subunit mRNA synthesis and loss of ßA-protein in rat gonocytes immediately after birth (8). This coincides with the onset of synthesis of the activin antagonist, follistatin, by gonocytes. In organ cultures of the 3 dpp rat testis, activin increased gonocyte numbers and inhibited gonocyte differentiation, whereas follistatin in concert with FSH accelerated the maturation of these germ cells to form spermatogonia.

The local production and actions of several TGFß superfamily members have also been demonstrated in the ovary (see Ref.9 for review). In the mouse, GDF-9 produced by oocytes is essential for follicular development past the primary follicle stage, and it regulates the expression of genes involved in several stages of folliculogenesis (10, 11). Activin has also been shown to be a potent regulator of FSH responsiveness in granulosa cells, which are known to produce both activin and activin receptor subunits (12).

Because roles for TGFß family members have been described in many organs, we sought to determine whether Bambi has the potential to serve a widespread function in regulating TGFß superfamily signaling. In our studies of spermatogenesis we had previously observed the discrete switch from synthesis of activin to follistatin in the transition of gonocytes to form spermatogonia (8). We reasoned that other mechanisms may exist to down-regulate TGFß signaling at the onset of spermatogenesis, and so we proceeded to specifically investigate the pattern of Bambi mRNA expression in the juvenile and adult rat testis. These observations led us to examine the potential for activin to modulate Bambi expression at the onset of spermatogenesis and to compare the cellular expression pattern in the testis with that in the ovary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and sample preparation
Sprague Dawley rats, ranging from birth [0 d postpartum (dpp)] to adulthood (60–90 dpp), and adult BALB/c male mice were obtained from Monash University Central Animal Services. The animals were killed by decapitation (rats) or cervical dislocation (mice) before tissue removal. All investigations conformed to the National Health and Medical Research Council/Commonwealth Scientific & Industrial Research Organization/Australian Agricultural Council Code of Practice for the Care and Use of Animals for Experimental Purposes and were approved by the Monash University standing committee on ethics in animal experimentation. Immediately upon removal, tissue samples for in situ hybridization analysis were placed in Bouin’s fixative for 5 h, then dehydrated and embedded in paraffin. Samples for RNA preparation were snap-frozen and stored at -70 C until use. Enriched fractions of pachytene spermatocytes (at least 86% pure) and round spermatids (at least 90% pure) were obtained from adult rats (13). Purity was verified by morphological assessment of thin sections of paraffin-embedded fixed cell preparations.

Testis fragment culture
Testis fragments from 3 d postpartum rat testes were cultured for 24 h as previously described (8). Fragments of approximately 1 mm3 were incubated in the presence of DMEM alone (control) or supplemented with activin A (100 ng/ml), follistatin (100 ng/ml), FSH (200 ng/ml; NIH S-17, NIADDK), or FSH plus follistatin. At the end of the culture period, the samples were snap-frozen and stored at -80 C for subsequent extraction of RNA. Three separate experiments were performed and analyzed by real-time PCR as described below.

Probe preparation and RNA analysis
RNA was prepared using the acid-phenol extraction method (14). A cDNA corresponding to bp 407–684 of the human nma sequence (accession no. U23070; hereafter referred to as hBambi) was prepared using RT-PCR from human testis RNA. This was used for Northern blot and in situ hybridization experiments (see below). RT was performed using 2 µg total RNA, 500 ng oligo(deoxythymidine)15 (Promega Corp., Madison, WI) and 200 U SuperScript II reverse transcriptase enzyme (Life Technologies, Inc., Grand Island, NY) in a 20-µl reaction according to the enzyme manufacturer’s guidelines. Block cycler PCR was performed using a Taq polymerase enzyme (Amersham Pharmacia Biotech, Piscataway, NJ) in a PerkinElmer 2400 block cycler (Norwalk, CT) with 10 pmol of each primer in a 20-µl reaction volume with the following cycling conditions: 1 cycle at 94 C for 5 min; 35 cycles at each 94, 58, and 72 C for 30 sec; and 1 cycle at 72 C for 5 min. The forward primer (F1) was 5'-tgcagctggagctctgcgc-3', and the reverse primer (R1) was 5'-gcacatgtcttcatgacagc-3'. A single product of the expected size resulted. It was cloned into pBluescript KS+ (Stratagene, La Jolla, CA) and sequenced (Wellcome Trust Sequencing Facility, Monash Medical Center) to confirm its identity. The PCR product was directly labeled with 32P using random primers with RTS Rad Prime DNA Labeling System (Life Technologies, Inc.) for use on Northern blots. Qualitatively identical results were obtained using an antisense 32P cRNA probe.

A second RT-block cycler PCR product was generated using rat testis RNA as the template and Platinum Pfx DNA polymerase with proof-reading activity (Life Technologies, Inc.) to determine the sequence of the full-length rat homolog. The primers were designed from sequences in the rat established sequence tag database corresponding to regions predicted to occur at the 5' and 3' ends of the Bambi coding sequence (accession no.: 5' end, gi8083136, bp 251–270; 3' end, gi7164471, bp 1208–1188). Noncoding sequence was included at both ends to assess concordance of the full-length rat coding sequence with that observed in other species. The forward primer was 5'-gcggggcgtcaatggatcgc-3', and the reverse primer was 5'-gaactcagaaggccttcaagg-3'. The product was cloned and sequenced in both directions as described above.

Real-time PCR was performed using RNA prepared from the cultured testis fragments. Two micrograms of total RNA from each sample were treated with deoxyribonuclease I (1 U; Ambion, Austin, TX) for 1 h at 37 C, and the enzyme was inactivated by heating to 90 C for 5 min. RT was performed as described above, and PCR samples were prepared using the Roche SYBR-Green DNA light cycler kit according to the manufacturer’s instructions. The F1 and R1 primers were used in the presence of 3 mM MgCl2 under the following conditions for 45 cycles of amplification and with amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (15) used to normalize samples. Primers were also used to measure the levels of inhibin {alpha} mRNA present in testicular fragments (15), as this mRNA has been shown to be elevated by FSH (16, 17, 18, 19). Melting curve analysis and agarose gel electrophoresis were used to monitor the production of a single product of the appropriate size and composition during the PCR. Negative controls, which contained water instead of sample RT, showed no amplification (data not shown).

Each experiment was analyzed in triplicate, and the data were analyzed by converting the relative amount of target mRNA in each treatment group to a value proportional to that measured for the control group (cultured in DMEM alone), so that this value is represented as the arbitrary unit of 1. The data are presented as the mean ± SE of the combined data from all three experiments, with errors less than 10%. Statistical significance was assessed by t test with Newman-Keuls multiple comparisons.

RNA blots contained 30 µg total RNA/sample separated on a 1.2% formaldehyde gel and transferred to a Nytran Super Charge membrane (Schleicher & Schuell, Keene, NH) following the manufacturer’s instructions. Hybridization was performed in Ultrahyb (Ambion) at 42 C for 16 h, and the membrane was washed with 0.1% standard saline citrate/0.1% sodium dodecyl sulfate at 42 C before exposure to x-ray film.

In situ hybridization using digoxigenin-labeled cRNAs was used to localize rBambi mRNA on rat testis sections using procedures previously described (20) with hybridization and washing temperatures up to 55 C and the incorporation of a ribonuclease wash. Both antisense and sense (negative control) cRNAs were used on each sample in every experiment for each set of conditions tested.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transcript characterization
Comparison of the rat Bambi (rBambi) nucleic acid and deduced amino acid sequences demonstrated a high level of identity with Bambi of other species. The rat and mouse deduced amino acid sequences show 97% identity, whereas the rat and human deduced amino acid sequences are 95% identical.

Northern blots identified a 1.8-kb transcript in all rat tissues examined from both 7 dpp and adult animals (Fig. 1Go), whereas a 1.5-kb transcript was observed in adult human testis RNA, the size corresponding to that reported for hnma (3) (data not shown). The 1.8-kb transcript was also detected in rat testis at all ages (0–90 dpp), with an additional 3 kb band detected in the d 30 and adult samples (Fig. 2AGo). Both bands were observed in RNA from enriched preparations of spermatocytes and round spermatids (Fig. 2BGo). A 1.8-kb transcript was also detected in the d 10 mouse testis, whereas additional 1.6- and approximately 2.2-kb bands were present in the adult mouse testis (Fig. 2BGo). The hBambi probe also recognized a 1.8-kb transcript in adult rat ovary, whereas the caput and cauda epididymis samples had barely detectable signals.



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  		FIG. 1. Nothern blot analysis demonstrates widespread tissue distribution of Bambi mRNA in d 7 and adult rats. Upper panels, Autoradiographs illustrating prominent Bambi mRNA signals at approximately 1.8 kb. The positions of 28S and 18S rRNA bands are indicated. Lower panels, Ethidium bromide gels show 28S and 18S RNA to indicate relative sample loading. H, Heart; SM, smooth muscle; Lu, lung; Sp, spleen; K, kidney; I, intestine; Li, liver; T7, 7 dpp testis; Bl, bladder; Tad, adult testis.

 


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  		FIG. 2. Northern blot analysis illustrates Bambi expression in the mammalian testis. Upper panels, Autoradiographs. Arrows indicate the positions of Bambi mRNA signals. The locations of 28S and 18S rRNAs are indicated. Lower panels, Ethidium bromide gels show 18S RNA to indicate relative loading between lanes of total RNA. A, Rat testis age series from date of birth (d 0) to adulthood (Ad). B: 1, Adult mouse testis; 2, d 10 mouse testis; 3, adult rat testis; 4, d 10 rat testis; 5, rat ovary; 6, rat spermatocytes; 7, rat spermatids; 8, rat cauda epididymis; 9, corpus epididymis; 10, caput epididymis.

 
Cellular localization in adult testis and ovary
In situ hybridization was used to examine specific cellular sites of rBambi expression in several testis samples. The signal was evident within all Sertoli cells on d 0 and 5 postpartum (Fig. 3GoGo, A and B) and within some adult Sertoli cells (Fig. 3EGoGo). In adult testis, the rBambi mRNA signal was most intense in germ cells from the intermediate/B spermatogonia through to late pachytene spermatocytes (stage XIV). A relatively less intense, but obvious, signal was detected in haploid round and early elongating spermatids to step 12, and a faint signal was observed in step 13–16 spermatids (Fig. 3GoGo, C, E, and F). No signal was detected in late elongating spermatids (steps 17–19), or in the samples hybridized with the sense cRNA that was used in each experiment (Fig. 3GoGo, A', B', C', and J'). Little or no discernable staining was present within the interstitial cell populations, and although difficult to assess with complete certainty due to the signal from adjacent cell types, there appeared to be staining in the cytoplasm of peritubular cells (Fig. 3EGoGo). These observations are summarized in Fig. 3FGoGo.



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  		FIG. 3. In situ hybridization showing cellular localization of Bambi mRNA within rat testis and ovary sections. A–J, Sections hybridized with antisense probe (labeled AS). A', B', C', and J', Sections hybridized with sense probe in the same experiment (labeled S). A–E, Rat testes at the indicated ages. D and F, Stage-specific differences in mRNA signal. Black open arrow, spermatocyte; white open arrow, round spermatid; black double arrowhead, elongating spermatid; white asterisk (d 0 and d 5; A, A', B, and B') and white filled arrow (adult testis; C and E), Sertoli cell; white arrow, gonocyte (A; d 0) or spermatogonium (B; d 5; E, type B). Int, Interstitium (C and E). F, Staging diagram to illustrate relative levels of signal intensity observed in cells of adult testis. Adapted from Russell et al. (37 ). G–J, Adult ovary sections. Black arrows, Granulosa cells; black double-headed arrows, oocyte; white asterisk and I, corpus luteum. Bar, 25 µm in A and B; 50 µm in C; 200 µm in G and H. (Figure is continued on next page.)

 


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  		FIG. 3A. Continued

 
In all follicles of the ovary, the rBambi signal was most intense within granulosa cells and in the internal layer of thecal cells (Fig. 3GoGo, G, H, and J). Variable staining intensity was evident within cells of the same follicle, but there was no obvious pattern of distribution of these cells. Other cells between the follicles and within the corpus luteum (Fig. 3GoGo, H and I) had a signal comparable to that observed in the thecal and granulosa cells (Fig. 3GoGo, F and G), whereas staining within oocytes was undetectable (Fig. 3GoGo, G and J).

Cellular localization and regulation in the developing testis
In the early postnatal rodent testis, Bambi signal was present in Sertoli cells, Leydig cells, and cells surrounding the developing cords (Fig. 3AGoGo). There was no apparent difference in the signal within these cell types between birth and 5 dpp (Fig. 3GoGo, A and B). In contrast to the somatic compartment, the Bambi mRNA signal was low to undetectable in the gonocytes at birth, but was readily observed in all germ cells present at 5 dpp. To correlate this change in Bambi expression within developing germ cells with our previous observation of a switch from activin to follistatin synthesis as gonocytes transform into spermatogonia (8), we employed testis fragment cultures to examine whether activin or follistatin affected the synthesis of Bambi mRNA during this time.

The relative levels of GAPDH mRNA measured by real-time PCR were used to normalize between samples in each experiment to ensure equal sample input per analysis and thereby enable quantitative comparison between different treatment groups with the same primer pair. To assess the validity of this approach, we first determined that there was no significant difference in the levels of GAPDH among the treatment groups. This indicated that changes in GAPDH expression did not occur in response to addition of exogenous factors (inter- and intraexperimental differences showed <1% variation; data not shown).

To examine our ability to assess the responsiveness of the testis fragments to exogenous factors, changes in the expression level of inhibin {alpha} subunit mRNA were next measured. The level of inhibin {alpha} subunit production was unaffected by the addition of activin A (1.12 ± 0.23) or follistatin (1.46 ± 0.25), compared with the DMEM only control level (set at 1; Fig. 4AGo). As predicted by results from previous studies, inhibin {alpha} mRNA levels were stimulated nearly 3-fold in the presence of FSH (2.80 ± 0.61; P < 0.001). The addition of follistatin to FSH-stimulated fragment cultures did not further change the level of inhibin {alpha} mRNA expression (3.14 ± 0.6; P < 0.001).



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  		FIG. 4. Real-time PCR analysis of d 3 rat testis fragment cultures demonstrates the suppression of Bambi mRNA production at 24 h of culture with activin A. The relative changes in Bambi expression in cultured testis fragments when treated with the indicated factors are shown. A, Analysis using inhibin {alpha} primers. B, Analysis using Bambi primers. Data are presented as the mean ± SE of three experiments. Distinct letters above each bar denote statistically significant differences between groups (P < 0.05).

 
The amount of Bambi mRNA present in testis fragments after 24 h of culture was significantly reduced by the addition of activin A (0.67 ± 0.09; P < 0.05) compared with DMEM only (Fig. 4BGo). The addition of follistatin alone (1.05 ± 0.13) or in combination with FSH (0.76 ± 0.12) had no effect on Bambi expression. There was no significant effect of FSH alone (1.05 ± 0.14) compared with either DMEM or activin A addition.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The findings presented in this study demonstrate that both testis and ovary are sites of significant expression of mRNA encoding the promiscuous TGFß signaling inhibitor Bambi, as demonstrated by Northern blot and in situ hybridization analysis. Furthermore, identification of Bambi mRNA in a variety of juvenile and adult rat tissues suggests that it is an important component of the complex network of mechanisms that regulate signaling by members of the TGFß superfamily.

In the developing testis, rBambi transcripts in germ cells are regulated to an extent that is easily observed by in situ hybridization during their maturation. The signal in the quiescent gonocytes at the center of the seminiferous cord at birth is weak to absent, but these cells and their progeny, the spermatogonia, contain a readily detectable hybridization signal 5 d later. The significance of this result is highlighted in the context of independent observations concerning the function and expression of TGFß family members at this interval. Our previous work has demonstrated that the activin ßA mRNA and protein are made by fetal rat gonocytes, and that the protein persists until about 3 dpp. Then, as the gonocytes transform into spermatogonia over the next few days, activin ßA protein disappears, and the synthesis of follistatin mRNA and protein is initiated in these cells (8). Thus, the loss of activin protein from within gonocytes corresponds to the onset of Bambi synthesis within these cells, an observation in accord with the suppression of Bambi mRNA levels by activin A measured in this study.

In vitro studies with 3 dpp rat testis fragments showed that although exogenous activin A supported an increase gonocyte numbers, the combination of follistatin plus FSH elevated the number of these cells that moved to the basement membrane to form spermatogonia (8). Bambi, like follistatin, may down-regulate activin and/or TGFß superfamily bioactivity at this crucial window of testis development. The data presented here support the concept that local synthesis of activin and its regulators is required to achieve temporal control of male germ cell differentiation, as activin within gonocytes may suppress their differentiation in an autocrine fashion. These observations in the rat model may be related to the finding in development of the Drosophila female germline, where expression of the Bmp2/4 homolog, decapentaplegic, is essential to maintain stem cell proliferation and its overexpression can block differentiation (21).

The presence of rBambi in a variety of germ cell types indicates that there is a requirement for regulating TGFß superfamily member action within or on germ cells. In the adult animal, spermatocytes contain the most intense signals, suggesting that these cells are likely to be the predominant sites of Bambi mRNA and protein synthesis. The distinct hybridization signal detected in type B spermatogonia and in round and early elongating spermatids reveals the possibility that a broad spectrum of germ cell types employs Bambi in local regulation of TGFß superfamily member bioactivity.

In addition to the 1.8-kb mRNA reported in the original analysis of hBambi expression in human tissue samples (3), a second transcript of 3-kb detected in the adult rat testis samples was identified in meiotic and postmeiotic germ cells. This is in accordance with the appearance of the second band in the rat testis samples taken between 20 and 30 dpp, the interval when the late meiotic and haploid germ cell populations emerge. Variant mRNAs are common in meiotic and postmeiotic germ cells (22), presumably reflecting the use of novel transcriptional machinery that employs cryptic splice sites and recognizes uncommon sites for poly(A)+ tail addition.

The results of in situ hybridization analyses demonstrate that rBambi mRNA is made in Sertoli cells of both immature and adult testes, apparently independent of their differentiation state. Quantitation of Bambi mRNA levels within individual cell types was not performed in this study, so there may be a change in the absolute amount of this mRNA made within the maturing Sertoli cells that we were unable to detect.

There are several known sites of TGFß superfamily member synthesis and action in the developing and adult testis, but we understand very little about how these interact (reviewed in Ref.23) and are regulated by hormones. The impact of TGFß and activin on germ cells and Sertoli cells in the developing testis has been shown in both coculture and organ culture systems (7, 8, 24, 25, 26). It is clear that, at least in the case of activin responsiveness, hormonal cues are important and vary with testis maturation (25, 27). The expression of BMP8a and BMP8b in germ cells of the adult testis has also been demonstrated (28), and a crucial role for these in spermatogenesis was revealed through analysis of the BMP8a knockout mouse, which has lesions in spermatogonial and spermatocyte populations (29). Other approaches uncovered a functional difference in the capacity of activin ßA and activin ßB gene products to support spermatogenesis. Replacement of the activin ßA-subunit-coding sequence with the activin ßB-coding region using the endogenous activin ßA promoter resulted in mice showing delayed onset of fertility in males and females, with an obvious lag in germ cell maturation during the first wave of spermatogenesis (30). This result, in which the expression of activin ßB protein in an activin ßA null mouse was able to compensate for the absence of activin ßA in several other functional aspects, emphasizes the importance of understanding the finer details of spatial and temporal regulation of TGFß superfamily member function. It is also interesting to note that up-regulation of Bambi mRNA in mouse fetal fibroblasts cultured with BMP4 has been observed (31), further highlighting the need to understand the plethora of signals that may be interacting to effect cell differentiation at the onset of spermatogenesis. Nagano and colleagues (32) recently reported that, in complete contrast to glial cell line-derived neurotrophic factor, activin and BMP4 each have a negative effect on the maintenance of stem cell potential in vitro. It is possible that establishment of the first stem cell population at the onset of spermatogenesis may require the suppression of differentiation signals, with the commitment to continue differentiation regulated by additional imputs.

Within the ovary of the adult rat, the age examined in this study, Bambi mRNA was readily detected in granulosa cells. Previous reports have documented in vitro and in vivo inhibin and activin subunit production in granulosa cells, with their synthesis shown to be regulated by TGFß and FSH (12, 33, 34, 35). Both TGFß and activin modulate the functional response of granulosa cells to FSH (12, 36).

It is striking that granulosa cells and Sertoli cells each show an age dependency in their responsiveness to the integration of these signals. Rat Sertoli cells at 9 dpp, but not 3 dpp, show increased proliferation in response to the combination of activin and FSH, but not to either of these alone (25). This has been attributed to discrete up-regulation of activin type II receptor mRNA synthesis on d 7–9 postpartum in rat Sertoli cells (27). The FSH and FSH plus TGFß stimulations of dimeric inhibin A production in ovaries of 8 dpp rats were observed only in preantral (primary and secondary follicles) (15). It will be interesting to ascertain whether these examples of changing responsiveness are attributable to regulated levels of Bambi protein production or function within granulosa and Sertoli cells.

Our findings have demonstrated the regulated expression of Bambi mRNA in male germ cells, which correlates well with our previous observation of a role for a discrete diminution in activin bioactivity at the time of gonocyte differentiation. In the adult testis, its widespread expression within the seminiferous epithelium indicates that it may regulate TGFß signaling at several stages of spermatogenesis. The predominant expression of Bambi in granulosa cells of the rat ovary corresponds to its localization to Sertoli cells of the testis and broadens its interest as a potential regulator of reproductive function. These data highlight the importance of modulated TFGß superfamily signaling within gonads that is required for the coordinated development and function of somatic and germ cells. A more detailed understanding of the expression patterns and functional interactions between TGFß superfamily members within the seminiferous tubule should heighten our understanding of the key events of gamete production.


   Acknowledgments
 
The rat cDNA sequence has been deposited in GenBank with accession no. AF387513.


   Footnotes
 
This work was supported by the National Health and Medical Research Council of Australia (Grants 973218 and 143792), the Wellcome Trust, and the Deutsche Forschungsgemeinschaft.

Abbreviations: BMP, Bone morphogenetic protein; dpp, days postpartum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Received December 10, 2002.

Accepted for publication May 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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